Dissecting the role of LMP1 (CAO) induced chronic inflammation in EBV associated carcinogenesis

Latent membrane protein 1 (LMP1) is the primary oncogene of Epstein-Barr virus (EBV), which is associated with various malignancies including nasopharyngeal carcinoma (NPC) – a tumour of epithelial origin. The NPC tissues are so heavily infiltrated with leukocytes that the tumour is sometimes also called lympho-epithelioma. In order to understand the mechanisms involved in LMP1 induced carcinogenesis (and inflammation-associated carcinogenesis in general), our lab has generated transgenic mice expressing an NPC variant of LMP1 (LMP1CAO) in the epidermis. LMP1 induced signalling pathways are activated in the pathological tissue, including NFκB, JNK, MAPK and p38 MAPK. In addition multiple proteins involved in proliferation and inflammation are upregulated including EGFR and its ligands, VEGF, and MMP9 amongst others. The skin of these mice, particularly the ears, develops a progressive inflammatory pathology from birth, initiating with hypervascularization (Stage1 (St1)) and hyperplasia (St2), increasing inflammation, leading to necrosis (St3), ulceration (St4), keratocanthoma (St5) and occasional carcinoma formation. The transgenic model permits an analysis of chronic inflammation as it leads to carcinoma, and the important factors involved in this. This thesis describes the experiments and the pre-clinical trials conducted to dissect the inflamed milieu internal of the pre-neoplastic transgenic tissue, with a focus on the immune cells, immune response, autoantigens, danger signals, oxidative stress and perturbed metabolic pathways. The transgenic tissue showed increased infiltration of mast cells, helper T-cells (CD4+), cytotoxic T-cells (CD8+/GranzymeB+) and Treg cells (CD4+/CD25+/FoxP3+). Inhibition of leukocyte recruitment using in vivo L-selectin inhibition not only resulted in slowed progression of the pathology but also could reverse the phenotype, suggesting a prognostic as well as therapeutic potential of L-selectin inhibition. Several inflammatory markers and cytokines, including STAT3, s100A9, CD30, CD30L, L-selectin, IL-3 and IP-10, were upregulated in the transgenic tissue compared to the controls. The role of a pro-inflammatory environment in the progression of the LMP1CAO induced pathology was investigated further by genetic removal of a chemokine decoy receptor D6 that resulted in accelerated progression of the pre-neoplastic as well as neoplastic pathology. Antibody deposition is a feature of this pathology. In order to identify the antigenic targets, various proteomic techniques were used. Immunoprecipitation experiments showed that the chitinase like proteins (CLPs) specifically Chi3L1, Chi3L3 and Chi3L4, are autoantigens in the LMP1CAO induced inflammation. Western blot and IHC analyses of the LMP1CAO tissue revealed that CLPs are expressed at an early stage of inflammation while active chitinases are expressed at the later stages. Moreover, an attempt was made to investigate the sera and biopsies from NPC patients for the expression and secretion of human CLPs, CHI3L1 and CHI3L2. In another approach, the T-cell/B-cell interaction (and thus Ig class switching) was disrupted through genetic elimination of CD40. The LMP1CAO/CD40KO mice showed delayed progression of the inflammatory phenotype during later stages, suggesting that Ig deposition is factorial in this process. Metabolic fingerprinting of the LMP1CAO tissue revealed deregulation of several metabolic pathways and suggested that “metabolic demand precedes the increased supply during pre-neoplasia”. Several metabolic changes indicative of increased cell proliferation were identified in the transgenic tissue, including upregulation of phospholipid metabolism intermediates, such as choline, phosphocholine, glycerophosphocholines and lysophosphatidic acid. A tumour specific glycolytic programme was operational in the transgenic tissue along with increased levels of glutamine, suggesting tumour-like energy consumption. The transgenic tissue was under oxidative stress indicated by increased levels of H2O2. Finally, in vivo administration of an anti-oxidant, N-acetylcysteine (NAC), arrested the inflammatory phenotype at very early stages, suggesting that oxidative stress is one of the inciting dangers/damages in the LMP1CAO induced inflammation. To summarize, the investigations presented herein have identified several candidates of diagnostic and therapeutic potential that are worth exploring further; not only in relation to the LMP1CAO induced carcinogenesis but also for various cancers in general